Propionibacterial cell lines for organic acid production

ABSTRACT

Microbial cell lines suitable for industrial-scale production of organic acids and methods of making and isolating such cell lines.

CLAIM OF PRIORITY

This application is a continuation application of and claims priorityunder 35 U.S.C. § 120 to U.S. application Ser. No. 16/443,554, filed onJun. 17, 2019, which will issue as U.S. Pat. No. 10,808,266, which inturn claims the benefit under 35 USC § 119(e) to U.S. Patent ApplicationSer. No. 62/686,463, filed on Jun. 18, 2018, the entire contents of eachof which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 19, 2020, isnamed 37942-0022002seq.txt and is 102,522 bytes in size.

TECHNICAL FIELD

The disclosure generally relates to microbial cell lines thatoverproduce organic acid and methods of making the same.

BACKGROUND

Organic acids refer to carbon-containing compounds having acidicproperties. Examples of organic acids include acetic acid, citric acid,gluconic acid, lactic acid, propionic acid, among many others. Becausethey are fully degradable, organic acids can be used in the productionof biodegradable polymers. They also have other important industrialapplications, including as food additives.

SUMMARY

The disclosure provides microbial cell lines suitable forindustrial-scale production of organic acids and methods of making andisolating such cell lines.

In one aspect, a method of making and isolating a microbial cell line isprovided, where the isolated microbial cell line overproduces an organicacid compared to the parental microbial cell line. The method usesserial passage of a parental strain in pH-controlled culture mediasupplemented with the organic acid, preferably in non-immobilizedculture, where the pH is controlled at a value above the pKa value ofthe organic acid. In some embodiments, the pH is preferably in the rangebetween about 5.5 and about 7.5, more preferably at or near neutral,between about 6.0 and about 7.0, and most preferably at about 7.0. Insome embodiments, the culture media is solidified. In some embodiments,the culture medium is supplemented with the organic acid in an amountsufficient to inhibit normal microbial cell growth, e.g., to reducedoubling rate or growth rate, e.g., by at least 5%, 10%, 20%, 30%, 40%,50%, or more. In some embodiments, the organic acid is supplemented at aprogressively increasing amount in successive iterations of the serialpassage. In some embodiments, the organic acid is supplemented at thesame amount in successive iterations of the serial passage. In someembodiments, the organic acid is propionic acid, lactic acid, aceticacid, or butyric acid. In some embodiments, the organic acid ispropionic acid, e.g., the culture media is supplemented with about1.0%-3.0% of propionic acid, e.g., about 3.0% of propionic acid. In someembodiments, the parental cell line is a wild-type organism. In someembodiments, the parental cell line is a microbial cell line is derivedfrom unicellular microbes.

In another aspect, a microbial cell line that overproduces an organicacid is provided, where the microbial cell line is made and isolatedusing serial passage in pH-controlled culture media supplemented withthe organic acid, where the pH is controlled at a value above the pKavalue of the organic acid, preferably in the range between about 5.5 andabout 7.5, more preferably at or near neutral, between about 6.0 andabout 7.0, and most preferably at about 7.0.

In another aspect, a microbial cell line that overproduces an organicacid is provided, where the microbial cell line has mutations thatprimarily alter, directly or indirectly, the structure, composition,and/or function of the cellular envelope. Preferably, the microbial cellline includes at least 2 genome mutations identified in Table 3 oranalogous mutations. More preferably, the microbial cell line includesall of the genome mutations identified in Table 3 or homologousmutations. In one embodiment, the microbial cell line includes mutationsin at least 2 genes identified in Table 3 or analogous mutationsthereto. In another embodiment, the microbial cell line includesmutations in all of the genes identified in Table 3 or their homologs(e.g., homolgous genes in another species described herein). In anotherembodiment, the microbial cell line includes a mutation in O-antigenligase domain-containing protein. In another embodiment, the microbialcell line includes a mutation in M18 family aminopeptidase. In anotherembodiment, the microbial cell line includes a mutation in amino acidpermease. In another embodiment, the microbial cell line includes amutation in adenine glycosylase.

The microbe can be any microbe that produces an organic acid. In oneembodiment, the microbe is from the genus Propionibacterium(Acidipropionibacterium), and more preferably the species P.acidipropionici. In another embodiment, the microbe is from the genusLactobacillus, and more preferably the species L. acidophilus. Inanother embodiment, the microbe is from the genus Acetobacter. Inanother embodiment, the microbe is from the genus Gluconobacter. Inanother embodiment, the microbe is from the genus Clostridium, and morepreferably the species C. butyricum. In some embodiments, the organicacid is propionic acid. In some embodiments, the organic acid is lacticacid. In some embodiments, the organic acid is acetic acid. In someembodiments, the organic acid is butyric acid.

Also provided herein are methods of producing organic acids using themethods and microbes described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows growth of wild-type and mutant P. acidipropionici on solidbuffered medium with or without addition of 1.0% PA, showing thephenotype of Strain 3-1.

FIG. 2 shows production of PA by a mutant strain of P. acidipropionici(Strain 3-1) relative to wild-type in bioreactors using bleachedAmerican Beauty Cake Flour (WFM as bolus). 1:200 inoculation, 1 Lworking culture, 5% (w/v) glucose equivalent WFM, 30° C., pH 7 (NaOH,5M).

DETAILED DESCRIPTION

The most common organic acids are carboxylic acids, whose acidity isassociated with the carboxyl group (—COOH). They are generally weakacids with pKa values between about 4-5. Propionic acid (“PA”), forexample, is a carboxylic acid with the chemical formula C₂H₅COOH orC₃H₆O₂. It is a colorless, oily, and pungent (think Swiss cheese andsweat) liquid and has physical properties between those of the smallercarboxylic, formic, and acetic acids, and the larger fatty acids. It hasa molecular weight of about 74.1 g/mol, and a pKa of about 4.9, whichmeans in a solution having a pH of about 4.9, half of the PA is in theprotonated (or undissociated), uncharged state (C₂H₅COOH), while theother half is in the deprotonated (or dissociated), negatively chargedstate (C₂H₅COO⁻), known as propionate or propanoate ion, which can formsalt or ester compounds. As the pH decreases (becoming more acidic),more PA is in the protonated, uncharged state; when the pH increases(becoming more basic), more PA is in the deprotonated, negativelycharged state.

Because PA inhibits the growth of mold and some bacteria at levelsbetween 0.1 and 1% (w/v), PA and its salts are used as a preservative inboth animal feed and human food (such as baked goods). In the UnitedStates, PA is “generally recognized as safe,” or GRAS, by the Food andDrug Administration when used as a food additive. It is also approvedfor use as a food additive in Australia, New Zealand, and the EU. Inaddition, PA is an important intermediate in the synthesis of otherchemicals, such as cellulose-derived plastics, pesticides, fruitflavors, perfume bases, and pharmaceuticals.

While they are widely distributed in nature, commercial production oforganic acids has generally relied on chemical synthesis because it ismore economically competitive. For example, PA is currently commerciallyproduced almost exclusively through petrochemical processes. As pricesfor crude oil and petrochemicals increase, along with the rapiddevelopment in the biotechnology field, the economic gap betweenmanufacturing costs of PA via chemical synthesis and via microbialfermentation is narrowing. Coupled with growing concerns about energyshortages and environmental pollution, there has been an increasinginterest in commercial-scale biosynthesis of organic acids such as PAfrom renewable resources.

Microbial production of organic acids by fermentation has been known andused for centuries. For example, Aspergillus niger and Yarrowialipolytica have been used to produce citric acid; Lactobacillus has beenused to produce lactic acid; Clostridium has been used to produce aceticacid; Aspergillus niger and Gluconobacter have been used to producegluconic acid.

Propionibacterium is the microorganism most often used in the productionof PA (as well as vitamin B₁₂ and Swiss cheese). Propionibacterium is agram-positive, non-motile, non-spore forming, rod-shaped, anaerobicgenus of bacteria that includes the species P. freudenreichii, P.acidifaciens, P. cyclohexanicum, P. australiense, P. acidipropionici, P.jensenii, P. thoenii, P. microaerophilum, P. olivae, P. damnosum, P.propionicum, P. acnes, P. avidum, P. granulosum, P. humerusii, and P.lymphophilum. For industrial PA production, the most commonly usedstrain is P. acidipropionici. (A proposal has been made to reclassifythe species within the genus Propionibacterium into three novel genera:Acidipropionibacterium, Cutibacterium, and Pseudopropionibacterium(Scholz & Kilian 2016). However, Propionibacterium acidipropionci andAcidipropionibacterium acidipropionici are still used somewhatinterchangeably.) The optimal pH and temperature for Propionibacteriumcell growth are about 6.0-7.0 and about 30-37° C., respectively (Ahmadiet al. 2017). Cell growth is inhibited in pH less than about 5.0,although fermenters started at neutral pH can reach pH 4.4 (Rehbergerand Glatz 1998). Ahmadi et al. provides an overview of PA production onseveral carbon sources by various species of Propionibacterium asreported in the literature (Ahmadi et al. 2017) and is incorporatedherein by reference.

PA can also be produced by other anaerobic bacteria, such as certainspecies of Anaerovibrio, Bacteroides, Clostridium, Fusobacterium,Megasphaera, Propionispira, Selenomonas, and Veillonella.

There are a number of fermentation pathways that convert carbon sourcesto PA through a series of enzymatic reactions. The primary fermentationpathway involved in PA production, especially in propionibacteria, isknown as the Wood-Werkman cycle, which produces propionate frompyruvate, the terminal product from glycolysis, and involves manyintermediates, including oxaloacetate, malate, fumarate, succinate,succinyl-CoA, methylmalonyl-CoA, and propionyl-CoA, and many enzymes,including oxaloacetate transcarboxylase, biotin-dependentcarboxytransferase, CoA transferase, fumarate hydrolase, lactatedehydrogenase, coenzyme B₁₂-dependent methylmalonyl-CoA mutase, malatedehydrogenase, and succinate dehydrogenase.

While most pyruvate is converted to PA/propionate during fermentation,some is converted to acetate. The acetate formation pathway involvesintermediates acetyl CoA and acetyl phosphate, and enzymes pyruvatedehydrogenase complex, phosphotransacetylase, and acetate kinase.

A number of carbon sources have been used for microbial PA production,including glucose, fructose, maltose, sucrose, xylose, lactose,glycerol, lactate, flour hydrolysate, molasses, whey, and a combinationthereof. A number of culture systems such as batch, fed-batch, andcontinuous fermentation have been used.

However, for commercial-scale microbial production of organic acids tobe economically viable, the fermentation process must be able to convertcarbon sources at a high yield (amount of organic acid production fromcarbon source, typically measured in g/g) and high productivity (rate oforganic acid production, typically measured in g/L·h).

Various fermentation technologies, including fed-batch, continuousculture, multi-stage, cell immobilization, and extractive fermentationsystems, have been explored to increase the yield of organic acidproduction. However, the modest increase in yield and productivity oftencomes is offset by a significant increase in production cost.

For example, coculture methods have been used to produce PA using wheyas feedstock (WO 85/04901; EP 0141642 A1). WO 85/04901 describes the useof Lactobacillus casei subspecies rhamnosus in the presence ofVeillonella cricetid to interconvert lactate to propionate via atwo-stage fermentation process. In the first stage, carbohydrates areconverted to lactic acid by L. casei; in the second stage, lactic acidis fermented to PA by V. cricetid. (The genera Lactobacillus andVeillonella both belong to the phylum Firmicutes, whereas the genusPropionibacterium belongs to the phylum Actinobacteria.) EP 0141642 alsodescribes the use of a mixed culture of lactic acid-producing bacteria(L. casei) and PA-producing bacteria (P. shermanii) to maximize thefermentation yield. The coculture systems of WO 85/04901 and EP 0141642are reported to be very productive in terms of PA production fromlactose, with final yields ranging from 20-100 g/L. However, suchcoculture systems have considerable implications for process parameters.For example, they suffer from a lack of control over the growth andmetabolic activity of each member of the system, which can lead tofailure of either member to grow or to contribute to formation of thedesired product. A lack of reproducibility is common with coculturesystems.

One major problem associated with microbial production of organic acidsis the strong inhibitory effect of the end product on cell growth andthe fermentation process, leading to low production yield andproductivity. Acid tolerance was assumed to be crucial to improving theyield and productivity of PA-producing strains (Rehberger and Glatz1998). The elevated inhibitory effect of PA at pH 4.5-5.0 as compared tolactic acid was attributed to the fact that at this pH range, about halfof PA (which has a pKa of about 4.9) would be present in theundissociated, protonated, and uncharged form, whereas lactic acid(which has a pKa of about 3.1) would mostly be in the dissociated,deprotonated, and charged form. It was assumed that because theundissociated acid could penetrate the cell wall and membrane moreeasily, more PA than lactic acid could get into the cell and exert itsinhibitory effect. Enhancement of acid tolerance was thus thought to bean effective strategy to alleviate end-product inhibition and improve PAproduction. Accordingly, attempts have been made to create “acidtolerant” mutants of propionibacteria under high PA and eitheruncontrolled or low pH conditions.

For example, adaptive evolution via serial passage has been used toobtain mutant P. acidipropionici with improved acid tolerance (Woskowand Glatz 1991; Zhu et al. 2010). Serial passage is a method of growingmicroorganisms such as bacteria in two or more iterations in artificialenvironments, often created in a laboratory setting, to generatespontaneous mutations in the microorganisms as they evolve over thecourse of the experiment to adapt to one or more new environmentalconditions designed for the experiment. For example, repeatedlysubjecting microbes to extreme acidic conditions will lead tospontaneous mutations that allow the microbes to adapt to or toleratesuch conditions.

In prior work, to create mutations that confer acid-tolerance, themutant P. acidipropionici strains were adapted to increasing PAconcentrations by repeated and serial transfers in selection mediacontaining increasing amounts of PA (from 0.5% to 5% (Woskow and Glatz1991) or 1.5 g/L to 20 g/L (Zhu et al. 2010)) over a period of one yearor longer. Importantly, in these experiments, pH in the selection mediahaving increasing amounts of PA was not controlled, presumably becauseit was assumed that the inhibitory effects on cell growth and PAproduction were caused by the acidity of PA.

P. acidipropionici mutant(s) with enhanced PA production has also beenobtained by immobilization and adaptation in a fibrous-bed bioreactor(Suwannakham and Yang 2005; Suwannakham 2005). The ability to obtainacid-tolerant mutant(s) in fibrous-bed bioreactor was attributed to thehigh cell density and viability maintained in the bioreactor anddistinct physiology and survivability of immobilized cells as a resultof their direct contact with each other and with a solid surface. Thehigher PA production was attributed in part to higher activity levels ofoxaloacetate transcarboxylase and CoA transferase in the mutant(s).Despite the higher PA yield, in the fibrous-bed bioreactor with highcell density, cell growth is limited. Moreover, fibrous-bed bioreactorsare expensive and not scalable, and their uses are limited tosmall-to-medium scale productions.

More recently, random mutagenesis strategies such as genome shufflinghave been used to accelerate directed microbial evolution. For example,Guan et al. reported the use of genome shuffling to generate anacid-tolerant mutant P. acidipropionici strain (Guan et al. 2012). Toobtain the strain, four successive rounds of genome shuffling viaprotoplast fusion were performed, and the acid-tolerant strain wasselected using media supplemented with increasing amounts of PA (from 5to 20 g/L). Again, pH in the selection media having increasing amountsof PA was not controlled, presumably because it was assumed that theinhibitory effects on cell growth and PA production were caused by theacidity of PA.

Subsequent analyses identified 24 proteins that significantly differedbetween the parental and shuffled strains (Guan et al. 2014). Thedetected proteins were reported to fall into four broad functionalclasses: cellular metabolism and energy production; DNA replication, RNAsynthesis, and translation; posttranslational modification, proteinfolding, and chaperones; and hypothetical proteins of unknown function.

In another study, genome shuffling was used to generate acid-tolerantmutant P. acidipropionici, P. intermedium, and P. jensenii strains (WO2017/055932 A2). Three successive rounds of genome shuffling wereperformed for each set of strains, each followed by selection ofcolonies from the acidic (pH 3) side of pH/PA gradient plates preparedusing agar culture media supplemented with 5 g/L of PA at either pH 3 orpH 6.5. Final individual recombinants were randomly selected afterserial dilutions in culture media plates and screened in a 96 well platecontaining 100 μ1 of culture media at pH 5 and 25 g/L of PA. The mutantstrains were reported to have enhanced yields of PA relative to nativePropionibacterium and other known derivative strains. Genomic analysesof one of the mutant P. acidipropionici strains identified a number ofmodified genes, including those encoding the ABC polar amino acidtransporter, the Cytochrome C biogenesis protein, the ABC multiple sugartransporter, the large subunit of ribosomal RNA, the long chain acyl-CoAsynthetase, and the cation diffusion facilitator. In addition, an extracopy of the whole ribosomal RNA gene and an extra copy of the argininedeiminase regulon (ArgR) with a point mutation were found in the mutantstrain.

Targeted metabolic engineering of propionibacteria has also been used toincrease PA production. These studies generally target enzymes involvedin pyruvate metabolism pathways to, for example, either inhibit theacetate formation pathway or enhance the PA formation pathway. Forexample, Yang and Suwannakham created engineered P. acidipropionicistrains with genes encoding acetate kinase (which catalyzes conversionof acetyl phosphate into acetate) and/or phosphotransacetylase (whichcatalyzes conversion of acetyl CoA into acetyl phosphate) knocked out,with the goal of eliminating or reducing acetate formation and therebyenhancing PA production (US 2011/0151529 A1; Suwannakham 2005).

Yang et al. created engineered P. acidipropionici and P. freudenreichiisubsp. shermanii strains transformed with propionyl-CoA:succinate CoAtransferase genes to increase PA production by overexpressionpropionyl-CoA:succinate CoA transferase, which catalyzes conversion ofpropionyl CoA into propionate (WO 2012/064883 A2). The resulting strainswere reported to have increased PA production and resistance to PA, aswell as resistance to acidic pH in general. The increased CoAtransferase activity is believed to increase carbon flux through the PAformation pathway over the acetate formation pathway.

The table below describes a list of genes that have been manipulatedusing recombinant DNA. These genes constitute conventional genetictargets where regulatory mutations might be expected to increase PAyields.

TABLE 1 Gene(s) Organism Effect Reference OtsA (trehalose P.acidipropionici Artificially over- Jiang et al. 2015 biosynthesis)expressed Several genes in P. jensenii Artificially over- Guan et al.2016 arginine deaminase and expressed glutamate decarboxylase systemsPropionyl- P. acidipropionici Artificially over- Wang et al. 2015CoA:succinate CoA P. shermanii expressed WO 2012/064883 A2 transferaseAcetate kinase P. acidipropionici Artificial knock out Suwannakham etal. 2006 Suwannakham 2005 US 2011/0151529 A1 Phosphotransacetylase P.acidipropionici Artificial knock out US 2011/0151529 A1

Targeted genetic engineering in propionibacteria, however, ischallenging. As an initial matter, the effect of acid alteration andstress on bacterial physiology is complex and not well understood,making it difficult to improve tolerance towards organic acids throughmanipulation of specific genes. Indeed, despite knowledge about theidentity of the intermediates and enzymes in the Wood-Werkman pathwaythat form PA in propionibacteria, genetic manipulations of the genes inthis pathway have not increased PA yields to a significant extent.

Moreover, the high GC content in propionibacteria makes it difficult toidentify the locations of individual genes and all of the coding regionsin the genome, which complicates genetic manipulation. In addition,there are only a small number of cloning vectors available forintroducing recombinant DNA into propionibacteria cells, which are knownto have low transformation efficiency. Selection of transformants isalso complicated by the ability of propionibacteria to quickly developspontaneous resistance to antibiotic markers.

In addition to these challenges, the use of recombinant DNA forproducing microbial cell lines is incompatible with the development ofan organic food ingredient such as PA. At least in the United States, PAor other organic acids produced by genetically engineered microbescannot be labeled as “organic” or “natural preservative,” which isespecially important in the food industry.Therefore, there remains aneed for new microbial strains suitable for industrial-scale productionof organic acids and methods of making and isolating such strains.

The toxicity of organic acids towards microbes is not well understooddespite its relevance in the food and chemical industries that usefermentation for organic acid production. Despite knowledge about theidentity of the intermediates and enzymes in the Wood-Werkman pathwaythat forms PA in propionibacteria, genetic manipulations of the genes inthis pathway have not increased PA yields to a significant extent. Onereason could be that these genes do not limit PA formation. Therefore,altering their sequence or expression would not change PA levels.Instead, it is argued here that other cellular targets control PAyields, but their identities could not be predicted based on currentknowledge. The unknown process is what limits PA formation. Since thisprocess is not known, the genes involved in this process cannot bepredicted.

Prior efforts in creating PA-resistant bacteria through serial passageor genome shuffling have generally used media with increasing amounts ofPA but either without pH control or at a pH significantly below the pKaof PA. This is based on the idea that toxicity, and thereforeresistance, arises from the concentration of the organic acid. However,this approach does not consider the mechanism of organic acid uptake bythe cell that involves the transporter system, which depends on thenature of the transporter and the membrane or envelope in which it islocated.

Organic acids are weak acids with pKa values generally between about4-5. The relationship between pH and pKa is described by theHenderson-Hasselbalch equation:

pH=pKa+log₁₀([A ⁻]/[HA])

wherein [HA] is the concentration of the protonated, undissociated, anduncharged weak acid, and [A⁻] is the concentration of the deprotonated,dissociated, and negatively charged conjugate base. In a typicalfermentation process, the pH of the microbial culture when the organicacid reaches maximum concentration is approximately at the pKa of theorganic acid without the use of a buffering agent. A solution having apH of about 4-5 is not that acidic relative to the known pH tolerance oforganic acid producing bacteria. Most of these bacteria do grow at pHvalues in this range, although the optimum pH for cell growth istypically about 6-7.

Intracellular transport of organic acids can be achieved throughdiffusion or through the action of membrane transport protein systemsdepending on whether the organic acids are charged or uncharged. Whenorganic acids are not deprotonated or dissociated, they are uncharged.In this state, they can diffuse across the cellular membranes withoutreliance on transport systems. Charged molecules, however, alwaysrequire a transport system to be translocated across membranes.

At a pH value that equals its pKa value, half of the organic acid is inthe protonated (or undissociated), uncharged form, while the other halfis in the deprotonated (or dissociated), negatively charged form. At pHvalues below their pKa values, organic acids would mostly be unchargedbecause their carboxyl groups would be protonated. At pH values abovetheir pKa values, organic acids would mostly be unprotonated ordissociated and therefore negatively charged.

At high concentrations of the organic acid, the pH is relatively low,and the organic acid would mostly be in the uncharged state and coulddiffuse into the cell in its acid form. This is the basis for priorefforts to isolate organic acid resistant microbes either without pHcontrol or at a pH significantly below the pKa of the organic acid. Theapproach in theory would generate cell lines with mutations thatproduced resistance due to diffusion-based organic acid cell entry. Itwas assumed that the uncharged organic acid would diffuse through thecell membrane into the cytoplasm and release protons due to therelatively alkaline pH inside the cell; the increase in intracellularacidity would inhibit cell growth and organic acid formation. In otherwords, it was assumed that organic acids in their uncharged statelimited their own production. Despite the published literature andpatents, in our experience, this approach does not generate resistantmicrobes effectively, and may require years of passage to work.

We hypothesized that it was not the acidity of the organic acid that wastoxic, as previously assumed by others. Rather, it was the deprotonated,negatively charged form or the neutral salt of the organic acid(propionate) that was toxic, and would be more effective as a selectionagent to recover spontaneous resistance mutations.

Unlike prior efforts, we hypothesized that the use of pH control at avalue above the pKa value of the organic acids to be produced, andpreferable at least 1 unit above, would ensure that most of the organicacids remain in a charged and deprotonated form. In this form, theywould remain dependent on protein transport systems for intracellularuptake. This would avoid recovery of cell lines with mutations thatproduced resistance due to diffusion-based organic acid cell entry, ifsuch mutations could be discovered.

Specifically, the process used was serial passage of the startingmicrobial cell line (usually but not necessarily a wild-type) infree-cell (i.e. non-immobilized or planktonic) culture in abacteriologic culture medium supplemented with organic acid of interestin an amount that is sufficient to inhibit normal microbial growth(either in progressively increasing amounts or the same amount for allpassages) under conditions of continued pH control at a specific pH thatis above the pKa value of the organic acid. The pH is controlled at avalue above the pKa value of the organic acid, preferably in the rangebetween about 5.5 and about 7.5, more preferably at or near neutral,between about 6.0 and about 7.0, and most preferably at about 7.0.Although the present examples describe the use of Propionibacterium,other microbes can be used that are fermentative organisms that excreteorganic acids, e.g., Lactobacillus, Acetobacter, Gluconobacter, orClostridium. The organic acid used can be, e.g., PA, lactic acid, aceticacid, or butyric acid. In some embodiments, the microbe is from thegenus Propionibacterium (Acidipropionibacterium), and more preferablythe species P. acidipropionici, and the organic acid is PA. In someembodiments, the microbe is from the genus Lactobacillus, and morepreferably the species L. acidophilus, and the organic acid is lacticacid. In some embodiments, the microbe is from the genus Acetobacter orthe genus Gluconobacter, and the organic acid is acetic acid. In someembodiments, the microbe is from the genus Clostridium, and morepreferably the species C. butyricum, and the organic acid is butyricacid.

Using our method of serial passage with pH control, we were able tocreate and isolate a new microbial strain having increased organic acidproduction compared to the parental strain in less than two weeks, muchfaster than using the conventional serial passage method described inWoskow and Glatz 1991, which generally takes at least one year. Ourmethod is also much less complex and more easily scalable than otherrandom mutagenesis methods such as genome shuffling and cellimmobilization in a fibrous-bed bioreactor or targeted geneticengineering. Organic acids produced by mutant cell lines created andisolated using serial passage with pH control can be labeled as“organic” or “natural preservative,” which is especially important inthe food industry.

The same method of serial passage with pH control can be used to makeand isolate a variety of microbes, including but not limited topropionibacteria, lactobacilli, acetic acid bacteria, and clostridia,that overproduce a number of organic acids, including but not limited toPA, lactic acid, acetic acid, and butyric acid. All charged moleculesdepend on transport systems and their associated membranes/envelopes forfunction. Alterations in these cellular components would achieve thesame outcome as described here for propionate for other organic acids.

The same selection method (i.e., using bacteriologic culture mediumsupplemented with organic acid of interest in an amount that issufficient to inhibit normal microbial growth under conditions ofcontinued pH control at a pH that is above the pKa value of the organicacid) can be used in screening microbial libraries generated from genomeshuffling or other random mutagenesis methods for isolates that exhibitincreased organic acid tolerance and production.

Using this pH control method, we were able to target unique mechanismsfor resistance that depended on transport and/or unpredictableintracellular targets including those involved in regulation andmetabolism. Genome resequencing was then used to identify the criticalgenes through their mutational changes that caused the geneticresistance to high concentrations of organic acids.

The resulting mutations generally affected cellular envelope functions,as shown in Table 2.

TABLE 2 ENVELOPE AND ASSOCIATED CATEGORIES ENVELOPE FUNCTIONS:Transporters/membrane proteins (10 affected ORFS): Major facilitatorsuperfamily proteins, amino acid permeases, hypothetical membraneprotein, LemA membrane protein, intramembrane metalloprotease, AAAATPase, sodium-proton antiporter Gain-of-function in penicillin-bindingprotein and amino acid permease Cell wall/peptidoglycan synthesis:Penicillin-binding proteins, O-antigen ligase domain- containingproteins (many mutations) ENVELOPE MODIFYING FUNCTIONS:Oxidation/reduction: Flavin reductase, alpha/beta hydrolase, pyruvatecarboxylase, MocA oxidoreductase, protophyringen oxidase, KGD Glycosyltransferases/hydrolases: Glycosyl transferase, glycosyl hydrolase,adenine glycosylase

These mutations primarily altered the structure and composition andfunction of the cellular envelope, which consists of the cell wall andmembrane(s), including the cytoplasmic membrane. A complete list of themutations identified is provided in Table 3. We did not see anymutations in genes that have been targeted for metabolic engineering andmanipulated using recombinant DNA as previously reported (see Table 1).Mutations in multiple genes appear to be required to produce the mutantphenotype (such as increased growth in media supplemented with organicacid and/or overproduction of organic acid compared to the startingmicrobial cell line). This is in direct contrast to prior knowledgewhere single genes were manipulated to try to change PA yields.

In accordance with the present invention, other conventionalmicrobiology, molecular biology, recombinant DNA, and biochemicaltechniques may be used. Such techniques are fully explained in theliterature and within the skill of the art. The invention will befurther described in the following examples, which do not limit thescope of the methods and compositions of matter recited in the claims.

EXAMPLE S Example 1

Isolation of Strain 3-1

A P. acidipropionici (ATCC 25562) was grown to high cell density in 10mL M24+2.0% glucose media. Serial dilutions of this culture (10⁰ to10⁻³) were then plated on solid M24+2.0% glucose media, solidified withagar, supplemented with 1.0%, 2.0%, and 3.0% (w/v) PA, all neutralizedto pH 7.0 using sodium hydroxide. Cells were also plated on solidM24+2.0% glucose media with no additional PA.

After a 5-day anaerobic incubation at 30° C., colony growth at thedifferent PA concentrations was assessed. Three colonies grew on the 3%PA plate plated with undiluted cells; no colony grew on the 3% PA platesplated with diluted cells. The three colonies were isolated andre-streaked onto no-PA, 2.0% PA, and 3.0% PA plates (all neutralized topH 7.0 using sodium hydroxide), along with freshly grown wild-type P.acidipropionici cells.

After a second 5-day anaerobic incubation at 30° C., colony growth atthe different PA concentrations was again assessed. All three isolates,but not wild-type, were able to grow on the 1.0% PA plate (FIG. 1). Onlyisolate #1 was able to grow on the 2.0% PA and 3.0% PA plates. Thisisolate was named strain 3-1 (“3” denotes 3.0% PA, and “1” denotesisolate #1). Isolate #1 was inoculated into 5 mL liquid M24+2.0% glucosemedia and grown to high cell density, and frozen permanents of thesecells were made.

After the phenotype of resistance to 3.0% PA on solid media wasconfirmed for strain 3-1, PA production in 10 mL batch cultures and 1 Lbioreactor cultures of this strain was compared to its parental P.acidipropionici (ATCC 25562) cells by HPLC in a broad range of media andcultivation conditions.

Strain 3-1 was deposited under the name NFS-2018 on Jul. 10, 2019, inthe American Type Culture Collection (10801 University Blvd. Manassas,Va. 20110-2209) and assigned Accession Number ATCC PTA-125895).

Example 2

PA Production by Strain 3-1 and Wild-Type P. acidipropionici

Wild type P. acidipropionici (ATCC 255562) and strain 3-1 werecultivated from a frozen permanent at 30° C. under anaerobic conditionin M24 medium supplemented with 2% glucose. The cells were sub-culturedevery 48 hr into fresh M24 medium starting at 10 mL then at 50 mL to useas seed for the 1 L bioreactor vessels.

For preparation of wheat flour medium, 75 g of American cake flour wasadded to 1 L of ddH2O in a sterile 2 L flask while mixing. One mL ofEnzenco alpha-amylase and 500 mL of 50 ppm of CaCl2 was added to themixture to hydrolyze the cake flour. The pH was adjusted to 6.0 byadding 5 mL of 5M NaOH and the temperature was held at 90° C. for 1hour. The mixture was allowed to cool then incubated at 37° C.overnight. After the overnight incubation, the temperature was raised to60° C. and pH adjusted to 7.0 by adding 2 mL of 5M NaOH. To releaseglucose, 1 mL of Enzenco glucoamylase, 0.05 g of protease, 0.4 g ofMgSO4, and 10 g of Ohly KAT yeast extract were added to the mixturewhile stirring. The mixture was held at 60° C. for 2 hours. The mixturewas allowed to cool then added to a glass-jacketed bioreactor vesselthen sealed. Before autoclaving, the pH was calibrated.

Fermentations were performed at 1 L volumes in the 3 L bioreactorvessels. The temperature was maintained at 30° C., the pH was maintainedat 7.0 using 5M NaOH, and cultures were agitated at 200 rpm. 3 mL offiltered sterile trace element solution was added to the bioreactorbefore inoculation. The glucose concentration was determined using a YSI2900 analyzer. The 1 L of wheat flour medium was seeded with 5%inoculum. Samples were removed every 24 hours for PA analysis on theHPLC. The results are shown in FIG. 2.

Both strain 3-1 and the parental wild-type strain reached maximum PAconcentration at about 120 hours. The maximum concentration of PAproduced by strain 3-1 is about 36 g/L, compared to about 30 g/L by theparental wild-type strain.

Additional experiments were carried out under 5-6 different conditions,3-4 times each, to compare PA production by strain 3-1 and wild-type P.acidipropionici. Results similar to those shown in FIG. 2 were obtained.There is a minimum of 15% increase in PA production by strain 3-1compared to the wild-type after 60 hours of culturing.

Example 3

Genomic Analyses of Strain 3-1

Genome resequencing of strain 3-1 was used to identify the criticalgenes through their mutational changes that caused the geneticresistance to high concentrations of organic acids.

65 loss of function mutations in 29 genes were identified. The mutationsgenerally affected cellular envelope functions, as shown in Table 2.These mutations primarily alter the structure and composition andfunction of the cellular envelope, which consists of the cell wall andmembrane(s), including the cytoplasmic membrane. A complete list of themutations identified in strain 3-1 is provided in Table 3.

TABLE 3 STRAIN 3-1 GENOME MUTATIONS SEQ ID Coordinates ORF NO. ChangeNon- synonymous 130744- ASQ49_RS00690 1 Arg → His 130746 class ISAM-dependent methyltransferase 130744- ASQ49_RS00695 2 Thr → Pro 130746MFS transporter 130748 ASQ49_RS00690 1 Pro → Leu class I SAM-dependentmethyltransferase 130752 ASQ49_RS00695 2 Arg → Gly MFS transporter181601 ASQ49_RS00915 3 Insertion (no (80% LemA family proteinframeshift) confidence) 181607- ASQ49_RS00915 3 Gln → Leu 181609 LemAfamily protein 240311 ASQ49_01155 4 Pro → His Flavin reductase 240440ASQ49_01155 4 Ala → Val Flavin reductase 281222 ASQ49_RS01330 5 Trp →STOP Hypothetical protein (BLAST hit to MFS transporter) 344598ASQ49_RS01635 6 Thr → Pro MFS transporter 525954 ASQ49_RS02385 7 Ala →Glu glycosyl transferase family 1 548143- ASQ49_RS02475 8 Ala → Gly548147 Hypothetical protein Gly → Leu (Strong BLAST hits to O-antigenligase and membrane protein) 548153- ASQ49_RS02475 8 Ala → Val 548156Hypothetical protein Gly → Leu (Strong BLAST hits to O-antigen ligaseand membrane protein) 548162 ASQ49_RS02475 8 Ala → Val Hypotheticalprotein (Strong BLAST hits to O-antigen ligase and membrane protein)558158- ASQ49_RS02520 9 His → Gly 558160 O-antigen ligasedomain-containing protein 558181 ASQ49_RS02520 9 Gln → His O-antigenligase domain-containing protein 558228 ASQ49_RS02520 9 Ala → ThrO-antigen ligase domain-containing protein 558252 ASQ49_RS02520 9 Ser →Pro O-antigen ligase domain-containing protein 558258 ASQ49_RS02520 9Ser → Pro O-antigen ligase domain-containing protein 558266ASQ49_RS02520 9 Arg → Leu O-antigen ligase domain-containing protein558273 ASQ49_RS02520 9 Ser → Ala O-antigen ligase domain-containingprotein 558279 ASQ49_RS02520 9 Gln → Glu O-antigen ligasedomain-containing protein 558282 ASQ49_RS02520 9 Glu → Gln O-antigenligase domain-containing protein 558288- ASQ49_RS02520 9 Leu → Pro588290 O-antigen ligase domain-containing protein 558291- ASQ49_RS025209 Glu → Val 558293 O-antigen ligase domain-containing protein 558306ASQ49_RS02520 9 Pro → Ala O-antigen ligase domain-containing protein558308- ASQ49_RS02520 9 Thr → Ser 558310 O-antigen ligasedomain-containing protein 562835 ASQ49_RS02535 10 Val → Leu O-antigenligase domain-containing protein 562840 ASQ49_RS02535 10 Ala → ValO-antigen ligase domain-containing protein 562843 ASQ49_RS02535 10 Gly →Ala O-antigen ligase domain-containing protein 566353 ASQ49_RS02550 11Lys → Gln (*50% penicillin-binding frequency) protein 566356ASQ49_RS02550 11 Ala → Ser (*50% penicillin-binding frequency) protein618017- ASQ49_RS02820 12 Glu → Ala 618019 Phosphotransferase 738302ASQ49_RS03340 13 Thr → Ala Alpha/beta hydrolase 742073 ASQ49_RS03360 14Ile → Leu Hypothetical protein (BLAST hits to intramembranemetalloprotease) 1176596 ASQ49_RS05220 15 Ala → Val gfo/Idh/MocA familyoxidoreductase 1279986 ASQ49_RS05630 16 Ile → Val Alpha/beta hydrolase1331356 ASQ49_RS05840 17 Gly → Ser Amino acid permease 1331366ASQ49_RS05840 17 Arg → His Amino acid permease 1521847 ASQ49_RS06625 18Thr → Ala Hypothetical protein (BLAST hits to protoporphyrinogenoxidase) 1816621 ASQ49_RS07985 19 Ser → Ala Adenine glycosylase 1816687ASQ49_RS07985 19 In-frame Adenine glycosylase insertion (1 amino acid)1817191 ASQ49_RS07985 19 Gly → Glu Adenine glycosylase 1817202ASQ49_RS07985 19 Glu → Ala Adenine glycosylase 1854503 SQ49_RS08150 20Lys → Arg Hypothetical protein (BLAST hit to sodium-proton antiporter)1854520 SQ49_RS08150 20 Ile → Met Hypothetical protein (BLAST hit tosodium-proton antiporter) 2679601 ASQ49_12020 21 His → Aspmultifunctional oxoglutarate decarboxylase/ oxoglutarate dehydrogenasethiamine pyrophosphate- binding subunit/ dihydrolipoyllysine- residuesuccinyltransferase subunit (kgd) 2927020 ASQ49_RS13125 22 In-frameAmino acid permease insertion (4 amino acids) 2927030 ASQ49_RS13125 22Gly-Ser → Amino acid permease Ala-Ala 2928883 ASQ49_RS13130 23 Asn → TyrHypothetical protein (glycosyl gydrolase family) 3517645 ASQ49_RS1596524 Thr → Arg (*50% M18 family frequency) aminopeptidase 3517646ASQ49_RS15965 24 Thr → Ser (*50% M18 family frequency) aminopeptidase3517648 ASQ49_RS15965 24 Ser → Thr (*50% M18 family frequency)aminopeptidase 3517649 ASQ49_RS15965 24 Ser → Gly (*50% M18 familyfrequency) aminopeptidase 3517652 ASQ49_RS15965 24 Ser → Tyr M18 familyaminopeptidase 3517655 ASQ49_RS15965 24 Ser → Asn M18 familyaminopeptidase FRAMESHIFTS 558244 ASQ49_RS02520 9 O-antigen ligasedomain-containing protein 558246 ASQ49_RS02520 9 O-antigen ligasedomain-containing protein 2867178 ASQ49_RS12835 25 AAA ATPase FRAMESHIFTREPAIRS 448285 ASQ49_RS02075 26 DUF1116 domain- containing protein561527 ASQ49_JRS02530 27 Glycosyl transferase 900222 ASQ49_RS03980 28acetyl-CoA carboxylase biotin carboxyl carrier protein subunit 919056ASQ49_RS04070 29 Penicillin-binding protein 1330401 ASQ49_RS05840 17Amino acid permease 1330407 ASQ49_RS05840 17 Amino acid permease

Mutations in these genes (or their homologues in other species describedherein) likely confer genetic resistance to high concentrations oforganic acids by altering the membrane transport protein systems and/orpreviously unknown intracellular targets involved in regulation and/ormetabolism.

Multiple mutations in the same gene imply that the gene is veryimportant for the trait and required multiple changes to contribute tothe trait. Noticeably, several genes had three or more mutations, whichmay indicate their critical roles in limiting organic acid formation.They include genes encoding: O-antigen ligase domain-containing protein(15 mutations in ASQ49_RS02520; 3 mutations in ASQ49_RS02535; and 3mutations in ASQ49_RS02475 (hypothetical protein with strong BLAST hitsto O-antigen ligase and membrane protein)); M18 family aminopeptidase (6mutations in ASQ49_RS15965); amino acid permease (4 mutations inASQ49_RS05840); and adenine glycosylase (4 mutations in ASQ49_RS07985).

REFERENCES

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1. A method of making and isolating a microbial cell line that producesan organic acid comprising serial passage in non-immobilized culture ofa parental cell line in a culture medium supplemented with the organicacid in an amount sufficient to inhibit normal microbial cell growth,wherein pH of the culture medium is controlled at a value above the pKavalue of the organic acid, and wherein the microbial cell line isolatedoverproduces the organic acid compared to the parental cell line.
 2. Themethod of claim 1, wherein the culture media is solidified.
 3. Themethod of claim 1, wherein the organic acid is supplemented at aprogressively increasing amount in successive iterations of the serialpassage.
 4. The method of claim 1, wherein the organic acid issupplemented at the same amount in successive iterations of the serialpassage.
 5. The method of claim 1, wherein the pH of the media iscontrolled at a value within the range of about 5.5-7.5.
 6. The methodof claim 5, wherein the pH of the media is controlled at a value withinthe range of about 6.0-7.0.
 7. The method of claim 6, wherein the pH ofthe media is controlled at about 7.0.
 8. The method of claim 1, whereinthe parental cell line is wild-type.
 9. The method of claim 1, whereinthe microbial cell line is derived from unicellular microbes.
 10. Themethod of claim 9, wherein the microbial cell line is derived from thegenus Propionibacterium, Lactobacillus, Acetobacter, Gluconobacter, orClostridium.
 11. The method of claim 9, wherein the microbial cell lineis derived from P. acidipropionici.
 12. The method of claim 1, whereinthe organic acid is propionic acid, lactic acid, acetic acid, or butyricacid.
 13. The method of claim 12, wherein the culture media issupplemented with about 1.0%-3.0% of propionic acid.
 14. The method ofclaim 13, wherein the culture media is supplemented with about 3.0% ofpropionic acid.
 15. A microbial cell line produced according to themethod of claim
 1. 16. A microbial cell line having at least 2 mutationsidentified in Table 3, preferably wherein the microbial cell line isderived from the genus Propionibacterium, Lactobacillus, Acetobacter,Gluconobacter, or Clostridium.
 17. The microbial cell line of claim 16,wherein the microbial cell line has all of the mutations identified inTable
 3. 18. The microbial cell line of claim 16, having loss offunction mutations in at least two genes encoding a protein identifiedin Table 3 or their homologs.
 19. The microbial cell line of claim 18,wherein the microbial cell line has loss of function mutations in all ofthe genes encoding the proteins identified in Table 3 or their homologs.20. A microbial cell line having at least one loss of function mutationin genes encoding proteins selected from the group consisting of (1)O-antigen ligase domain-containing protein, (2) M18 familyaminopeptidase, (3) amino acid permease, and (4) adenine glycosylase.21. The microbial cell line of claim 20, wherein the at least one lossof function mutation is in the gene encoding O-antigen ligasedomain-containing protein.